Copper monooxygenases are essential to human health. They catalyze the biosynthesis of neuropeptide hormones and catecholamine neurotransmitters, and thus have special significance with respect to the health of neuroendocrine pathways. Their lack of activity in patients with Menkes disease (caused by mutation of the ATP7A copper pump) leads to severe neurological dysfunction, while peptidylglycine monooxygenase is elevated in certain cancers where it stimulates tumor proliferation via over production of growth hormones. Amidated peptides are also critical regulators of appetite suppression, and therefore are central players in preventing obesity. In addition to their biomedical impact, these enzymes are highly significant from a biochemical perspective since they react via novel chemical mechanisms. Understanding these mechanistic paradigms within the broader context of copper biochemistry is the subject of the proposal. There are three specific aims. Aim 1 will test a new hypothesis that the intramolecular pathway for electron transfer is bifurcated, with structurally determined gating mechanisms in place to optimize ET efficiency. Experiments will probe the enzymatic response to mutation of putative pathway residues, the structural contributions of the electron transferring H-center to gating, and how structural perturbation and/or mutation of CuH-binding residues influences the catalytic properties of the M-center. Aim 2 will explore how substrate binding activates the enzyme to bind and reduce molecular oxygen using a unique chemical marker for substrate triggering in the form of a perturbed CO binding state at the catalytic M-center. New approaches using FTIR, XAS and NMR are targeted at understanding the origins of substrate triggering. Aim 3 will search for catalytic oxygen intermediates using innovative protocols to maximize the build-up of intermediates which include (i) Increasing the rate of formation of the intermediate using hyperoxygenated solutions (ii) carrying out reactions in the presence of substrates predicted to stabilize the Cu(I)-dioxygen/Cu(II)-superoxo complex and (iii) slowing the rate of decay by use of deuterated substrates, radical-destabilizing substrate analogues, or variants with compromised ET pathways. Intermediates trapped by these methods will be studied by advanced spectroscopic and computational tools. Expected outcomes of the proposal are a better understanding of the unique ET machinery of this class of monooxygenases, how the binding of substrates influences oxygen reactivity, and a description of the reactivity of their catalytic intermediates in terms of chemical and electronic structure.